Clostridium difficile-derived membrane vesicles induce the expression of pro-inflammatory cytokine genes and cytotoxicity in colonic epithelial cells in vitro

- C. difficile strains secrete spherical membrane vesicles (MVs) during in vitro culture.
- MVs of C. difficile ATCC 43255 contain a total of 262 proteins.
- C. difficile MVs stimulate the expression of pro-inflammatory cytokine genes in Caco-2 cells.
- C. difficile MVs induce cytotoxicity in Caco-2 cells.
- C. difficile MVs are important nanocomplexes that may contribute to bacterial pathogenesis.

Clostridium difficile is the most common etiological agent of antibiotic-associated diarrhea in hospitalized and non-hospitalized patients. This study investigated the secretion of membrane vesicles (MVs) from C. difficile and determined the expression of pro-inflammatory cytokine genes and cytotoxicity of C. difficile MVs in epithelial cells in vitro. C. difficile ATCC 43255 and two clinical isolates secreted spherical MVs during in vitro culture. Proteomic analysis revealed that MVs of C. difficile ATCC 43255 contained a total of 262 proteins. Translation-associated proteins were the most commonly identified in C. difficile MVs, whereas TcdA and TcdB toxins were not detected. C. difficile ATCC 43255-derived MVs stimulated the expression of pro-inflammatory cytokine genes, including interleukin (IL)-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 in human colorectal epithelial Caco-2 cells. Moreover, these extracellular vesicles induced cytotoxicity in Caco-2 cells. In conclusion, C. difficile MVs are important nanocomplexes that elicit a pro-inflammatory response and induce cytotoxicity in colonic epithelial cells, which may contribute, along with toxins, to intestinal mucosal injury during C. difficile infection.